1. Field of the Invention
This invention relates to the field of preserving living cell cultures. More particularly a method is provided which allows surface-attached cell cultures to be cooled, stored and later revived without greatly disturbing their adherence to the original surfaces upon which they were attached and without destroying their capability for being revived as living cell cultures.
2. Description of Prior Observations
Polge, Smith and Parkes, Nature, London 164:666 (1949) discovered that bull spermatozoa can be preserved for long periods of time by freezing suspended cells in the presence of 5-10% glycerol. Since their discovery many workers have used modifications of this technique for the preservation of living cells of various types. Subsequently, a technology developed which has been aimed at improving the efficiency with which suspensions of living cells can be revived after storage in the frozen state.
While Shimada and Ashina, Cryobiology 9:51-56 (1972) have studied the freezing and thawing of surface-attached living cells, they were principally interested in microscopically observing individual HeLa cells which were attached to cover-slips during the freezing the thawing processes in the absence of the chemicals usually employed for long term storage of cells in the frozen state. The storage aspects of frozen cells were not studied by Shimada and Ashina, rather, they thawed the cells within minutes after freezing, at different rates and with and without ice crystals "seeding". The physical effects of these treatments on the morphology of the cells was observed.
Variables which have been studied for storing suspended cells include incorporation of different chemical components into the storage medium (See Vos and Kaalen, Cryobiology 1:249-260 (1965) and Ashwood-Smith,Warby, Becker and Connor, Cryobiology 9:311 (1972)), the concentration of these components, the rate at which the temperature is lowered during the initial cooling process, the temperature at which cells are stored and the rate at which cells are warmed during revival. Scherer and Hoogasian, Proc. Soc. Exp. Biol. Med. 87:480-487 (1954), using glycerol in their storage medium, did some of the first experimental work to determine optimal conditions for storing suspended cells derived from cell cultures. Later, dimethyl sulfoxide (DMSO) was found useful for storing suspended cells at low temperatures (See Farrant, Nature, London 205:1284-1287 (1965). Glycerol and DMSO are, at the present time, probably the most generally used chemicals employed in the various media utilized for storing suspended cells derived from cell cultures (see the discussion by Paul, Cell and Tissue Culture, 4th Ed., pp. 308-315, The Williams and Wilkins Company, Baltimore (1970) and that by Wasley and May, Animal Cell Culture Methods, pp. 137-144, Blackwell Scientific Publications, Oxford and Edinburgh (1970)). About 5% to about 15% concentrations of glycerol or DMSO are now most commonly used, although Scherer and Hoogasian successfully used glycerol at concentrations up to 30%. The latter workers also reported successful storage of both HeLa and L cell cultures after rapid cooling; however, slow cooling at about one centigrade degree per minute is now generally favored and practiced. In fact, Meryman, Annals of the New York Academy of Sciences, 85:503-509 (1960) stated that, except for mammalian erythrocytes, "rapid freezing does not appear to work for the majority of animal tissues". Meryman further stated "we must abandon for the time being the rapid freezing approach which, whether because of intracellular crystal growth or some other undetermined mechanism, simply does not work". Cells of different kinds seem to vary in their sensitivity to rapid cooling, as well as to other variables in the storage process. Slow cooling, at the rate of about one centigrade degree per minute, gives satisfactory results for storing many kinds of suspended cells derived from cell cultures, by the processes now practiced, and is the method now most generally used.
With minor variations, freezing and storage of cultured cells is presently accomplished in the following manner: Cells adhering to and growing on vessel surfaces are washed with physiological saline and are detached from such surfaces by mechanical means (shaking or scraping) and/or by the use of enzymes and a chelating agent (such as EDTA). The cell suspension is usually centrifuged, the liquid phase containing the enzymes or chelating agent discarded (some workers eliminate this step, but it is recommended for optimal results) and the cells re-suspended in a liquid suitable for freezing (usually this contains about 5% to about 15% DMSO or glycerol). The cells are kept at 0.degree.-4.degree.C while a sample is counted in a hemacytometer and then the volume of the liquid is adjusted to give the desired cell concentration (usually about 2.sup.. 10.sup.6 cells/ml). Measured volumes of the cell suspension are placed into multiple ampoules (cell suspensions must, of course, be continually agitated during this period to keep them in a homogeneous suspension). The ampoules are then sealed, (usually with a hot torch), frozen slowly (special equipment is usually required) and maintained at a storage temperature of -70.degree.C or below until needed. The frozen cell suspensions are then thawed rapidly, centrifuged, the liquid phase containing the storage medium is discarded, the cells are resuspended by agitation and the cells placed into appropriate culture vessels to grow. Cultures are divided after cell crowding occurs and, when a sufficient number of cells have developed in a sufficient number of the appropriate type of culture vessels, they are used for specific purposes such as virus experimentation, isolation of viruses from clinical specimens, titration of viruses, virus production, or further cell propagation. Summarizing, the methods for cell freezing and storage now commonly used involve the following manipulations: detachment from the culture surface by mechanical agitation, scraping or enzyme treatment, centrifugation to remove the enzyme, resuspension in storage medium, continuous agitation, cell counting, adjustment of the cell concentration, dispensing into ampoules, sealing the ampoules, slow freezing, thawing, re-centrifugation to remove the storage medium, continuous agitation, cell counting, adjustment of the cell concentration, dispensing into ampoules, sealing the ampoules, slow freezing, thawing, re-centrifugation to remove the storage medium, resuspension by agitation, and dispensing into new culture vessels for cell growth. These manipulations, in addition to being numerous and likely to shock or damage cells, are time consuming, require enzymes, and/or chelating agents, require a centrifuge, require a device for controlled freezing, require special sealable ampoules for storage and require the use of new culture vessels for growth of the revived cells. When done expertly under the best of circumstances recovery rates of up to 95% viable cells have been attained. Figures less than this are frequently obtained because it is difficult to achieve uniformly optimal performance at every one of the many steps involved. Poor recovery of viable cells in some cases is probably due to a peculiar susceptibility of certain cell types to one or more of these manipulations.